Vaccines based on streptokinase

ABSTRACT

The present invention provides a vaccine to treat or prevent mastitis in a bovine species, comprising an immunologically effective amount of a plasminogen-activating streptokinase protein produced by Streptococcus uberis. The present invention further provides a method of treating or preventing mastitis in a bovine species comprising vaccinating a cow with an immunologically effective amount of a plasminogen-activating streptokinase protein produced by S. uberis. The present invention further provides an isolated plasminogen-activating streptokinase protein produced by S. uberis, or an immunogenic fragment thereof.

This application is the U.S. national stage of PCT/GB93/00110, filedJan. 18, 1993, which International Application claims priority GreatBritain application No. 9201013.1 filed Jan. 17, 1992.

This invention relates to vaccination against diseases caused bypathogens, and more particularly to vaccination against mastitis.

To cause clinical mastitis in the bovine udder a bacterium must eithergrow within the gland at a rate sufficient to avoid removal in thesecretion or must colonise normal secretory and/or ductular tissues.More virulent strains of bacteria may resist phagocytic killing despitethe presence of large numbers of polymorphonuclear leucocytes. Certainspecies of bacteria are known to produce haemolytic and/or cytolytictoxins which may have a role in the pathogenesis of the disease.

To date, vaccines to protect the mammary gland from clinical mastitishave attempted to promote more efficient phagocytosis and killing ofbacteria (by the production of opsonising antibody) or to inactivatetoxic products (by the production of neutralising antibody).

Streptococcus uberis is a common cause of bovine mastitis responsiblefor around 20% of all clinical cases in the UK (Bramley and Dodd 1984).The ability of this organism to infect the lactating mammary gland isdependant on its ability to grow in the secretion and avoid phagocytosisby bovine neutrophils (Leigh et al 1990).

The majority of nitrogen in bovine milk is present in the form ofprotein (Aston 1975) and, in the absence of proteolysis, bacterialgrowth in milk is limited by the lack of free amino acids. This ishighlighted by the dependence of the lactic streptococci onextracellular, caseinolytic proteinases for growth in milk (Mills andThomas 1981). The ability of bacteria to grow in mastitic milk isenhanced by the presence of the caseinolytic enzyme plasmin (Marshalland Bramley 1984). The transformation of plasminogen to plasmin requiresplasminogen activators which are known to occur in blood plasma andanimal tissues (Collen 1980). Certain streptococci are capable ofproducing streptokinase which activates plasminogen to plasmin but nopreviously isolated streptokinase activates bovine plasminogen.

According to one aspect of the present invention, there is provided avaccine for use to treat or prevent a disease in a vertebrate, thevaccine comprising an antigenic entity and a carrier, the antigenicentity being adapted to cause, following vaccination of the vertebratewith the vaccine, an immune response generating antibodies which inhibita factor from a pathogen which, directly or indirectly, causes breakdownof protein in the vertebrate wherein said breakdown enhances growth ofthe pathogen.

Antibodies which "inhibit" the factor are those which diminish to auseful extent the ability of the factor to cause the said breakdown ofthe vertebrate protein. Preferably, for any given interaction between anindividual antibody and a molecule of the factor, the said ability isreduced to zero. Suitably, the antibodies are secreted into theenvironment where the factor acts. Thus, in the case of vaccines againstmastitis, the antibodies should be secreted into the milk.

Streptococci are responsible for certain types of dental caries, thus,in the case of dental caries, the antibodies should be secreted into thesaliva or present in the mucous membranes associated with the gums andlining of the mouth.

Suitable carriers and adjuvants etc for formulating the antigen entityinto a vaccine are known.

Pharmaceutically acceptable carriers may, for example, be liquid mediasuitable for use as vehicles to introduce the antigenic entity into thevertebrate. An example of such a carrier is saline solution. Theantigenic entity may be in solution or suspended as a solid in thecarrier.

The vaccine formulation may also comprise an adjuvant for stimulatingthe immune response and thereby enhancing the effect of the vaccine.Convenient adjuvants for use in the present invention include, forexample, aluminium hydroxide and aluminium phosphate.

The vaccines of the present invention may be administered by anyconventional method for the administration of vaccines including oraland parenteral (e.g. subcutaneous or intramuscular) injection. Thetreatment may consist of a single dose of vaccine or a plurality ofdoses over a period of time.

By "treating or preventing" the disease we mean ameliorating an existingor future disease to a useful extent, and including reducinginflammation to a useful extent.

According to a second aspect of the present invention, there is provideda method of treating or preventing a disease in a vertebrate comprisingvaccinating the vertebrate with a vaccine as above.

We believe that the vaccines and methods in accordance with the presentinvention work by inhibiting a factor from a pathogen which directly orindirectly causes breakdown of protein in the host, such that free aminoacids are not generated and the pathogen cannot then grow sufficientlyquickly for the disease to persist.

The factor may directly or indirectly activate plasminogen in milk tocause proteolysis of milk proteins. In the case of vaccination againstmastitis, the factor may be a bacterial streptokinase capable ofactivating plasminogen in milk. Suitably, it may be a streptokinaseproduced by Streptococcus uberis. However, streptokinases from otherstreptococci may also activate plasminogen in milk, for example astreptokinase produced by Streptococcus dysgalactiae is a suitableplasminogen activator. Alternatively, the factor may be a bacterialprotease capable of causing hydrolysis of milk proteins, for example aStaphylococcus aureus protease, a number of which have been reported(see references).

The vaccine of the invention comprises any entity which causes aninhibitory immune response directed to the pathogen's factor discussedabove. The entity need not be the whole, native factor. Antigenicfragments may be used, especially those identified by hydrophilicityplots as being hydrophilic, as may modified forms of the factor whichare functionally inert, ie they do not cause the breakdown of protein inthe host, but which can be used to raise inhibitory antibodies againstthe factor.

Therefore, when the antigenic entity of the invention is a protein,preferably a bacterial protease, more preferably a streptokinase, theterm includes any variants and fragments of the protein which are usefulto prepare antibodies which will specifically bind the said protein ormutant forms thereof lacking the function of the native protein. Suchvariants and fragments will usually include at least one region of atleast five consecutive amino acids which has at least 90% homology withthe most homologous five or more consecutive amino acids region of thesaid protein. A fragment is less than 100% of the whole protein.

It will be recognised by those skilled in the art that an antigenicpolypeptide of the invention may be modified by known proteinmodification techniques. These include the techniques disclosed in U.S.Pat. No. 4,302,386 issued 24 Nov. 1991 to Stevens, incorporated hereinby reference. Such modifications may enhance the immunogenicity of theantigen, or they may have no effect on such immunogenicity. For example,a few amino acid residues may be changed. Alternatively, the antigenicentity of the invention may contain one or more amino acid sequencesthat are not necessary to its immunogenicity. Unwanted sequences can beremoved by techniques well known in the art. For example, the sequencescan be removed via limited proteolytic digestion using enzymes such astrypsin or papain or related proteolytic enzymes.

Alternatively, polypeptides corresponding to antigenic parts of theprotein may be chemically synthesised by methods well known in the art.These include the methods disclosed in U.S. Pat. No. 4,290,944 issued 22Sep. 1981 to Goldberg, incorporated herein by reference.

Peptides may be synthesised by the Fmoc-polyamide mode of solid phasepeptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433and references therein. Temporary N-amino group protection is affordedby the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage ofthis highly base-labile protecting group is effected using 20%piperidine in N,N-dimethylformamide. Side-chain functionalities may beprotected as their butyl ethers (in the case of serine threonine andtyrosine), butyl esters (in the case of glutamic acid and asparticacid), butyloxycarbonyl derivative (in the case of lysine andhistidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4'-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethylacrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalisingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivative are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversedN,N-dicyclohexyl-carbodiimide/1-hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used are ethanedithiol, phenol, anisole and water,the exact choice depending on the constituent amino acids of the peptidebeing synthesised. Trifluoroacetic acid is removed by evaporation invacuo, with subsequent trituration with diethyl ether affording thecrude peptide. Any scavengers present are removed by a simple extractionprocedure which on lyophilisation of the aqueous phase affords the crudepeptide free of scavengers. Reagents for peptide synthesis are generallyavailable from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.Purification may be effected by any one, or a combination of, techniquessuch as size exclusion chromatography, ion-exchange chromatography and(principally) reverse-phase high performance liquid chromatography.Analysis of peptides may be carried out using thin layer chromatography,reverse-phase high performance liquid chromatography, amino-acidanalysis after acid hydrolysis and by fast atom bombardment (FAB) massspectrometric analysis.

Thus, when the antigenic entity of the invention is a protein, thisincludes a class of modified polypeptides, including syntheticallyderived polypeptides or fragments of the original protein, having commonelements of origin, structure, and immunogenicity that are within thescope of the present invention.

Antibodies to the factor may be administered to the host to providepassive immunity, although this will usually be less desirable thanimmunising with a vaccine of the invention.

According to a third aspect of the present invention there is provided astreptokinase which is capable of activating mammalian plasminogen inmilk. Preferably, the streptokinase is or is substantially the same as astreptokinase produced by Streptococcus uberis.

A fourth aspect provides a nucleic acid sequence encoding thestreptokinase isolated from at least most of the genome sequence inwhich the sequence is found in nature. In other words the nucleic acidsequence is not claimed in the form in which it has previously existed.Thus, the nucleic acid sequence of the invention includes the nucleicacid sequence when that sequence has been cloned into a bacterialvector, such as a plasmid, or into a viral vector that may be harbouredby a bacteriophage provided that such clones are in isolation fromclones constituting a DNA library of the relevant chromosome.

It is possible to obtain the nucleic acid sequence that encodes thestreptokinase from the amino acid sequence of the streptokinase byutilising methods well known in the art. By using a stretch of aminoacid sequence from the protein, specific DNA probes can be synthesised,according to known methods, that will hybridize with the mRNA and DNAencoding the protein.

The gene may comprise the promoter and/or other expression-regulatingsequences which normally govern its expression and it may compriseintrons, or it may consist of the coding sequence only, for example acDNA sequence.

The nucleic acid sequence includes any variation which is (i) usable toproduce a protein or a fragment thereof which is in turn usable toprepare antibodies which specifically bind to the protein encoded by thesaid gene or (ii) an antisense sequence corresponding to the gene or toa variation of type (i) as just defined. For example, different codonscan be substituted which code for the same amino acid(s) as the originalcodons. Alternatively, the substitute codons may code for a differentamino acid that will not affect the activity or immunogenicity of theprotein or which may improve its activity or immunogenicity. Forexample, site-directed mutagenesis or other techniques can be employedto create single or multiple mutations, such as replacements,insertions, deletions, and transpositions, as described in Botstein andShortle, "Strategies and Applications of In Vitro Mutagenesis,"Science,229: 193-1210 (1985), which is incorporated herein by reference. Sincesuch modified genes can be obtained by the application of knowntechniques to the teachings contained herein, such modified genes arewithin the scope of the claimed nucleic acid sequence.

Moreover, it will be recognised by those skilled in the art that thegene sequence (or fragments thereof) can be used to obtain other DNAsequences that hybridise with it under conditions of high stringency.

Such DNA includes any genomic DNA. Accordingly, the gene of theinvention includes DNA that shows at least 55 percent, preferably 60 percent, and most preferably 70 percent homology with the gene of theinvention, provided that such homologous DNA encodes a protein whichcauses an inhibitory immune response directed to the pathogen's factoras described above.

"Variations" of the gene include genes in which relatively shortstretches (for example 20 to 50 nucleotides) have a high degree ofhomology (at least 50% and preferably at least 90 or 95%) withequivalent stretches of the gene of the invention even though theoverall homology between the two genes may be much less. This is becauseimportant active or binding sites may be shared even when the generalarchitecture of the protein is different.

Hereinafter, the term "gene" will be used to embrace all such variationsand fragments.

The gene or variation thereof may be used, when included in a suitableexpression sequence, to prepare an antigenic protein or fragment thereofusable in accordance with the present invention.

A fifth aspect of the invention provides a method of producing the saidprotein by expressing a corresponding nucleic acid sequence in asuitable host cell or by amino acid synthesis.

Thus, the nucleic acid of the invention may be used in accordance withknown techniques, appropriately modified in view of the teachingscontained herein, to construct an expression vector, which is then usedto transform an appropriate host cell for the expression and productionof an antigenic polypeptide of the invention. Such techniques includethose disclosed in U.S. Pat. No. 4,440,859 issued 3 Apr. 1984 to Rutteret al, U.S. Pat. No. 4,530,901 issued 23 Jul. 1985 to Weissman, U.S.Pat. No. 4,582,800 issued 15 Apr. 1986 to Crowl, U.S. Pat. No. 4,677,063issued 30 Jun. 1987 to Mark et al, U.S. Pat. No. 4,678,751 issued 7 Jul.1987 to Goeddel, U.S. Pat. No. 4,704,362 issued 3 Nov. 1987 to Itakuraet al, U.S. Pat. No. 4,710,463 issued 1 Dec. 1987 to Murray, U.S. Pat.No. 4,757,006 issued 12 Jul. 1988 to Toole, Jr. et al, U.S. Pat. No.4,766,075 issued 23 Aug. 1988 to Goeddel et al and U.S. Pat. No.4,810,648 issued 7 Mar. 1989 to Stalker, all of which are incorporatedherein by reference.

The gene of the invention may be joined to a wide variety of other DNAsequences for introduction into an appropriate host. The companion DNAwill depend upon the nature of the host, the manner of the introductionof the DNA into the host, and whether episomal maintenance orintegration is desired.

Generally, the gene, preferably as cDNA, is inserted into an expressionvector, such as a plasmid, in proper orientation and correct readingframe for expression. If necessary, the DNA may be linked to theappropriate transcriptional and translational regulatory controlnucleotide sequences recognised by the desired host, although suchcontrols are generally available in the expression vector. The vector isthen introduced into the host through standard techniques. Generally,not all of the hosts will be transformed by the vector. Therefore, itwill be necessary to select for transformed host cells. One selectiontechnique involves incorporating into the expression vector a DNAsequence, with any necessary control elements, that codes for aselectable trait in the transformed cell, such as antibiotic resistance.Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus), plant cells,animal cells and insect cells.

Those vectors that include a replicon such as a procaryotic replicon canalso include an appropriate promoter such as procaryotic promotercapable of directing the expression (transcription and translation) ofthe genes in a bacterial host cell, such as E. coli, transformedtherewith.

A promoter is an expression control element formed by a DNA sequencethat permits binding of RNA polymerase and transcription to occur.Promoter sequences compatible with exemplary bacterial hosts aretypically provided in plasmid vectors containing convenient restrictionsites for insertion of a DNA segment of the present invention.

Typical procaryotic vector plasmids are pUC8, pUC9, pBR322 and pBR329available from Biorad Laboratories, (Richmond, Calif., U.S.A.) and pPLand pKK223 available from Pharmacia, Piscataway, N.J., U.S.A.

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. The DNAsegment, generated by endonuclease restriction digestion as describedearlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNApolymerase I, enzymes that remove protruding, 3'-single-stranded terminiwith their 3'-5'-exonucleolytic activities, and fill in recessed 3'-endswith their polymerizing activities. The combination of these activitiestherefore generates blunt-ended DNA segments. The blunt-ended segmentsare then incubated with a large molar excess of linker molecules in thepresence of an enzyme that is able to catalyze the ligation ofblunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus,the products of the reaction are DNA segments carrying polymeric linkersequences at their ends. These DNA segments are then cleaved with theappropriate restriction enzyme and ligated to an expression vector thathas been cleaved with an enzyme that produces termini compatible withthose of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies Inc, New Haven, Conn., U.S.A.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either procaryotic or eucaryotic. Bacterial cells are preferredprocaryotic host cells and typically are a strain of E. coli such as,for example, the E. coli strains DH5 available from Bethesda ResearchLaboratories Inc., Bethesda, Md, U.S.A., and RR1 available from theAmerican Type Culture Collection (ATCC) of Rockville, Md., U.S.A. (NoATCC 31343). Preferred eucaryotic host cells include yeast and mammaliancells, preferably vertebrate cells such as those from a mouse, rat,monkey or human fibroblastic cell line. Preferred eucaryotic host cellsinclude Chinese hamster ovary (CHO) cells available from the ATCC asCCL61 and NIH Swiss mouse embryo cells NIH/3T3 available from the ATCCas CRL 1658.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used. With regard to transformation ofprocaryotic host cells, see, for example, Cohen et al, Proc. Natl. Acad.Sci. USA, 69: 2110 (1972); and Sambrook et al, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989). Transformation of yeast cells is described in Sherman etal, Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor,N.Y. (1986). The method of Beggs, Nature, 275: 104-109 (1978) is alsouseful. With regard to transformation of vertebrate cells withretroviral vectors containing rDNAs, see, for example, Sorge et al, MolCell. Biol., 4: 1730-37 (1984); Graham et al, Virol, 52: 456 (1973); andWigler et al, Proc. Natl. Acad. Sci. USA, 76: 1373-76 (1979).

Successfully transformed cells, ie cells that contain a DNA construct ofthe present invention, can be identified by well known techniques. Forexample, cells resulting from the introduction of an expressionconstruct of the present invention can be grown to produce the proteinof the invention. Cells can be harvested and lysed and their DNA contentexamined for the presence of the DNA using a method such as thatdescribed by Southern, J. Mol. Biol., 98: 503 (1975) or Berent et al,Biotech., 3: 208 (1985). Alternatively, the presence of the protein inthe supernatant can be detected using antibodies as described below.

In addition to directly assaying for the presence of recombinant DNA,successful transformation can be confirmed by well known immunologicalmethods when the recombinant DNA is capable of directing the expressionof the protein. For example, cells successfully transformed with anexpression vector produce proteins displaying appropriate antigenicity.Samples of cells suspected of being transformed are harvested andassayed for the protein using suitable antibodies.

Thus, in addition to the transformed host cells themselves, the presentinvention also contemplates a culture of those cells, preferably amonoclonal (clonally homogeneous) culture, or a culture derived from amonoclonal culture, in a nutrient medium. Preferably, the culture alsocontains the protein.

Nutrient media useful for culturing transformed host cells are wellknown in the art and can be obtained from several commercial sources.

A sixth aspect of the present invention provides a vertebrate vaccinatedagainst the host factor. In the case of vaccination against mastitis,the vertebrate is a mammal known to be prone to having mastitis, such asa pig, cow, sheep or horse. At least in the case of these mammals beingvaccinated against mastitis by the methods of the invention where theantigen is related to bacterial streptokinase, such mammals can bedistinguished from non-vaccinated mammals (even those which have hadmastitis) by the presence of neutralising anti-streptokinase antibodies,since the disease does not normally cause a neutralising immuneresponse, at least not as far as streptokinase is concerned.

It is evident that vaccination against mastitis in accordance with thepresent invention can be used in conjunction with known therapies formastitis.

The vaccines and methods of the present invention will be discussed andexemplified with specific references to the disease mastitis, theantigenic entity of the vaccine being a streptokinase produced by thecausative organism Streptococcus uberis or an antigenically equivalententity. However, the invention relates to a general principle and may beapplied to other diseases in which a factor from that pathogen causesbreakdown of protein in the host.

Auxotrophic bacteria cannot synthesize certain essential amino acids andthese must be provided in the medium for these organisms to grow. Theamino acids present in milk are in three forms: free amino acids,non-protein peptides (defined as being soluble in 12% w/vtrichloroacetic acid) and polypeptides and proteins. Most amino acidsare present in protein form. Bacteria can readily utilise free aminoacids and, in some instances, can use amino acids in the form of smallpeptides. However, to obtain sufficient amino acids to attain highdensity bacterial growth, or to obtain essential amino acids which onlyoccur in the protein form, auxotrophic bacteria must have a system forproteolysis of milk proteins.

It has been found that Streptococcus uberis has a very specific aminoacid requirement, as determined by growth studies in a chemicallydefined medium. These requirements are not met by the amino acidspresent in the free form in bovine milk, nor by those present in thenon-protein nitrogen form in bovine milk (Aston 1975). It is wellestablished that S. uberis grows well in the bovine udder: about 300colony forming units (cfu) infused into the udder result in around 10⁵-10⁷ cfu/ml milk after 12 hours.

We believe that the vaccines and methods in accordance with the presentinvention where the antigenic entity is related to bacterialstreptokinase work by inhibiting the bacterial streptokinase so that thehost's plasminogen is not activated and thus free amino acids are notgenerated in the milk and growth of the bacteria is thereby inhibitedsufficiently quickly for the disease not to persist.

The vaccine may comprise a substantially pure or a non-pure preparationof Streptococcus uberis streptokinase. It may also comprise apreparation containing inactivated forms of the streptokinase.

The S. uberis streptokinase, or any other plasminogen activating factorsor bacterial proteases found to be suitable as vaccines againstmastitis, may be administered by various different routes when used as avaccine. The vaccine is administered so as to produce sufficientantibody in the secretion of the lactating mammary gland to neutralisebacterially-induced proteolysis. This will promote protection of themammary gland to infection by the bacteria.

Streptokinase produced by S. uberis is capable of activating bovine,equine and ovine plasminogen. The streptokinase may be purified fromculture filtrates of S. uberis by ammonium sulphate precipitationfollowed by molecular exclusion chromatography. S. uberis appears toproduce a single protein possessing plasminogen activating activity. Themolecular weight of the native molecule is approximately 57 kD, whereasthat of the purified protein by SDS PAGE is 29 kD. This suggests thatthe native molecule consists of a dimer of the 29 kD sub-unit, which isdissociated during electrophoresis in the presence of SDS and2-mercaptoethanol.

The native molecular weight is distinct from those observed forstreptokinase from either Lancefield group A (46.7 kD) or Lancefieldgroup C streptococci (47.2 kD). However, both of these molecules existas monomeric structures and are not dissociated during SDS PAGE (Huanget al 1989). Staphylokinase, a plasminogen activator from Staphylococcusaureus, has a subunit molecular weight of between 23 kD (Jackson andTang 1982) and 15.3 kD (Sako et al 1982) and has been shown to require adimeric structure for activity (Jackson et al 1981). This molecule hasnot been shown to activate bovine plasminogen, and is unlikely to bepresent in strains of Staphylococcus aureus which infect the bovinemammary gland.

Streptococcus uberis streptokinase shows some immunological crossreaction with antisera raised to streptokinnase from other streptococci,reflecting the presence of similar antigenic sites on the molecules.

A further aspect of the invention provides a process of causingproteolysis in milk comprising adding a streptokinase to the milkwhereby the streptokinase activates plasminogen to the caseinolyticprotein plasmin.

The importance of plasmin in Swiss-type cheese ripe has beendemonstrated (Ollikainen and Kivela 1989). In a study of proteolysisduring the ripening of Swiss-type cheese, it was found that thehydrolysis of β-casein by plasmin and the hydrolysis products, ₋₋-caseins and protease-peptones, are typical of Swiss-type cheese. Theproduction of ₋₋ -casein and protease-peptones is indicative of plasminactivity, and it was concluded that plasmin is an important, evenessential, enzyme for Swiss-type cheese ripening. Therefore, there is apotential role for streptokinase in the cheese ripening process, whereflavour of the final product is dependant upon the presence of peptides,amino acids and their derivatives.

Another potential use of the streptokinase is as a fibrinolytic agent inspecies which have plasminogen which is susceptible to activation. Otherstreptokinases are currently used for this procedure in human patientssuffering from thrombolytic disorders.

The following examples illustrate preferred aspects of the invention ina non-limiting manner, with reference to the accompanying drawings, inwhich:

FIG. 1 illustrates an elution profile of ammonium sulphate precipitatedproteins from culture filtrate of Streptococcus uberis;

FIG. 2 illustrates the separation of ammonium sulphate precipitatedproteins and high streptokinase activity fractions from molecularexclusion chromatography. Ammonium sulphate precipitated proteins (1).Low purity fraction (2). High purity fraction (3). Prestained molecularweight standards (Sigma) with native molecular weights of 180, 116, 84,58, 48.5, 36.5 and 26.6 kD (4).

FIG. 3A and 3B illustrates the hydrolysis of milk proteins byStreptococcus uberis in the presence and absence of bovine plasminogen.Streptococcus uberis strain 0140J (A and B) overlaid with agarosecontaining skimmed milk in the presence (A) and absence (B) of bovineplasminogen.

FIG. 4 illustrates the detection of caseinolytic activity followingactivation of plasminogen with streptokinase from a Lancefield group Cstreptococcus or S. uberis culture filtrate. Wells contain a mixture ofstreptokinase from a Lancefield group C Streptococcus (column 1), S.uberis culture filtrate (column 2), or phosphate buffered saline (column3) and either human (row H), rabbit (row R), porcine (row P), equine(row E) or bovine (row B) plasminogen. The row labelled PBS containedphosphate buffered saline in place of plasminogen; and

FIG. 5 illustrates SDS PAGE of bovine plasminogen following incubationin the presence of S. uberis culture filtrate. Tracks of bovineplasminogen in the presence of 2 μl of phosphate buffered saline (A), S.uberis culture filtrate diluted 1/1000 (B), 1/100 (C) and 1/10 (D) inphosphate buffered saline (PBS). Arrows indicate the position of theplasmin associated polypeptides. No protein bands were detectedfollowing electrophoresis of culture filtrate diluted 1/5 in PBS.Numbers indicate the position of proteins of known molecular weight.

EXAMPLE 1 Purification of Streptokinase from Culture Filtrates

Streptococcus uberis strain 0140J was used throughout the presentinvestigation. This strain was originally isolated from a case of bovinemastitis at the National Institute of Dairy Research, Shinfield,Reading, England.

Bacteria were stored at -20° C. in Todd Hewitt broth (THB) containing25% (w/v) glycerol. Cultures were initially grown for 18 h in 10 ml ToddHewitt broth at 37° C. A chemically defined medium (Leigh and Field1991) containing casein hydrolysate (1%, w/v) and glucose (1%, w/v) wasinoculated with this culture (10 μl per 500 ml medium) and incubated at37° C. for 18 h. The cells were removed by centrifugation (10,000 g; 20min) and filtration (0.45 μm pore size) of the resulting supernatant.Sodium azide was added to the culture filtrate to a final concentrationof 0.05% (w/v).

The purification of streptokinase from culture filtrates may also beachieved by an affinity chromatography system using immobilisedmonoclonal antibodies. (Harlow, E. & Lane, D. (1988) Antibodies. Alaboratory manual. Cold Spring Harbour (U.S.A.).)

Ammonium sulphate precipitation from culture filtrates, gel filtrationand detection of streptokinase activity

Streptococcus uberis strain 0140J was used. Saturated ammonium sulphatesolution was added to the cell-free culture filtrate to a finalconcentration of 38% (v/v). The mixture was stirred at 4° C. for 20 hand the resulting precipitate collected by filtration (0.45 μm poresize).

The precipitate was redissolved in 100 ml of distilled water containing0.05% (w/v) sodium azide and dialysed for 24 h against 2.0 l of the samediluent. The dialysate was concentrated ten fold in a stirred pressurecell (Amicon, Mass, U.S.A.) using a membrane with 10 kD exclusion limit(Filtron, Mass, U.S.A.).

A column (28×1000 mm) containing Sephadex G-75 (Pharmacia, Uppsala,Sweden) with an approximate packed bed volume of 500 ml was equilibratedwith phosphate buffered saline (PBS) at 4° C. Approximately 10 ml of theammonium sulphate precipitated, redissolved, dialysed and concentratedprotein solution was applied to the column and proteins eluted in PBS ata flow rate of 0.5-1.0 ml/min. Fractions (10 ml) were collected and 5 μlof each assayed for bovine plasminogen activating activity.

Preliminary experiments demonstrated that streptokinase activity wasprecipitated from cell-free culture filtrates by ammonium sulphate at aconcentration of 33-38% saturation (data not shown). A concentration of38% saturation was subsequently used for the precipitation ofstreptokinase from 2.5 l of cell-free culture filtrate. The precipitatewas redissolved, dialysed and concentrated prior to gel filtration.

The streptokinase activity eluted from the G-75 column as a singleactive peak with an apparent molecular weight of approximately 57 kD(FIG. 1). Fractions 21 and 22 were pooled (high purity streptokinase) aswere fractions 19, 20, 23 and 24 (low purity streptokinase) and storedat -70 C with no apparent loss of activity.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE)and protein staining

The purity of the pooled fractions containing streptokinase activity wasdetermined using the SDS PAGE and protein staining technique previouslydescribed.

The redissolved, dialysed and concentrated ammonium sulphate precipitatecontained 6 proteins with molecular weights ranging from 100 to 29 kD.The low purity fractions contained the 29 kD protein and trace amountsof the others whereas the high purity fractions contained only a singleband with a molecular weight of 29 kD (FIG. 2).

EXAMPLE 2 Detection of Plasminogen Activation by Agarose/Skimmed MilkOverlay

Plasminogen from rabbit, human, porcine, equine and bovine plasma wasobtained from Sigma Chemical Co. (Poole, Dorset, UK) and reconstitutedin sterile distilled water to a final concentration of 1.0 unit ml⁻¹.Streptokinase from a Lancefield group C Streptococcus was also obtainedfrom Sigma, reconstituted at a concentration of 1 mg ml⁻¹ in phosphatebuffered saline (pH 7.4). Plasminogen and streptokinase were stored at-70° C. and thawed only once prior to use.

Overnight cultures in THB were streaked onto Todd Hewitt agar andincubated at 37° C. for 18 h. Plates with isolated colonies wereoverlaid with 10 ml of molten agarose (10 μml⁻¹) containing NaCl (150mM), Tris/HCl (50 mM, pH 8.1), Oxoid skimmed milk (1 μl % v/v) andbovine plasminogen (10 μg ml⁻¹) and incubated at 37° C. Controls wereperformed using overlays identical to that above except that plasminogenwas omitted.

All five strains of S. uberis (0140J, EF20, ST10, C216, C197C) producedzones of caseinolytic activity in skimmed milk, bovine plasminogen,agarose overlays within 4 hours at 37° C. (FIG. 3A). There may be somestrains of S. uberis, however, which do not produce this activity: thesewill probably show reduced virulence for the bovine mammary gland. Nozones were detectable around isolated colonies in overlays in theabsence of bovine plasminogen (FIG. 3B).

EXAMPLE 3 Plasminogen Activation and detection of Plasmin

Equal volumes of plasminogen (1.0 unit ml-¹ from a variety of mammalianspecies) and S. uberis culture filtrate or streptokinase (1 μ g ml-¹)from a Lancefield group C Streptococcus were mixed and incubated at 37°C. for 45 min after which 10 μl pI was assayed for the presence ofplasmin by the detection of caseinolytic activity. Activity was detectedby diffusion from wells cut in skimmed milk agarose (as overlays abovecontaining no plasminogen) following 24 h incubation at 37°C. (FIG. 4).

Culture filtrates from S. uberis activated bovine and equine plasminogenbut the activity with the latter was only apparent after incubation ofthe skimmed milk agarose for around 18 h compared to 2 h when usingbovine plasminogen. This suggests either a lower activity of S. uberisstreptokinase for this substrate or the poor activity of the resultingplasmin molecule for bovine milk proteins. S. uberis culture filtratefailed to activate plasminogen from human, rabbit or pig. In contrast,streptokinase from the Lancefield group C Streptococcus activated humanplasminogen and showed a trace of activity towards equine plasminogenbut no activity towards that from rabbit, bovine or porcine plasma.Neither S. uberis culture filtrate nor streptokinase from the Lancefieldgroup C Streptococcus showed any caseinolytic activity in the absence ofplasminogen.

Thus, the streptokinase activity present in S. uberis culture filtratediffered from that isolated from Lancefield group E streptococci whichis reported to activate porcine plasminogen (Ellis and Armstrong 1971);S. uberis culture filtrate failed to activate this molecule (FIG. 4). Italso differed from similar activities from S. equisimilis and S.pyogenesboth of which activate human but not bovine plasminogen(Castellino 1979, Wulf and Mertz 1969) whereas streptokinase from S.uberis activates bovine but not human plasminogen. This is the firstreport of 15 the presence of plasminogen activating activity in S.uberis and the first report of a streptokinase which activates bovineplasminogen.

EXAMPLE 4 Conversion of Plasminogen to Plasmin and Detection byPolyacrylamide Gel Electrophoresis and Protein Staining

Bovine plasminogen (0.005 unit in a volume of 5 μl ) was mixed with 2μof S. uberis culture filtrate and incubated at 37°C. for 1 h. Sampleswere mixed with an equal volume of sample-buffer containing sodiumdodecyl sulphate (0.01% w/v) and 2-mercaptoethanol (0.2% v/v) and heatedat 65° C. for 5 min. Proteins were separated by electrophoresis (Laemmli1970) and detected by the staining procedure of Oakley et al (1980)(FIG. 5).

The plasminogen used during this investigation contained contaminatingproteins. The suppliers claim the plasmin contamination to be less than5% total protein (Sigma Chemical Co, Poole, UK). Bovine plasminogen hadan apparent molecular weight of 91.2 kD and this agrees with thecalculated molecular weight based on the amino acid sequence of thisprotein (Schaller et al 1985). Activation of plasminogen was achievedafter 60 min by a 1/10 dilution of S. uberis culture filtrate (FIG. 5).This resulted in the loss of the 91.2 kD protein band together withanother protein (48.5 kD). It is not clear whether the disappearance ofthe 48.5 kD protein was a result of the action of the culture filtrateor of the resulting plasmin activity. The disappearance of these twoproteins corresponded with the formation of three smaller polypeptideswith apparent molecular weights of 56.2, 28.8 and 25.4 kD.

Activation of bovine plasminogen by urokinase (a plasminogen activatorof human origin) is predicted to result in the formation of only twopolypeptides with molecular weights of 53.7 and 25.4 kD (Schaller et al1985). These probably correspond to the proteins at 56.2 and 25.4 kD,respectively. The predicted 53.7 kD polypeptide contains two potentialglycosylation sites (Schaller et al 1985) and this might account for thediscrepancy between the predicted and the observed values. This suggeststhat the S. uberis streptokinase acts in a similar fashion to urokinaseduring the activation of the bovine plasminogen molecule.

Since no protein bands were seen following electrophoresis of culturefiltrate at a concentration two fold higher than that used to achievetotal plasminogen activation, the presence of an additional polypeptide(28.8 kD) cannot be explained simply by the presence of bacterialproteins. The cumulative total of the molecular weights of the threepolypeptides observed following plasminogen activation during thisinvestigation shows a significant discrepancy from that of bovineplasminogen. One possible explanation for this is that the 28.8 kDpolypeptide is a product of the degradation of the 48.5 kD protein whichcontaminates the plasminogen preparation and is depleted duringactivation. The determination of the molecular weights of the productsof the activation of purified plasminogen by purified S. uberisstreptokinase will resolve this discrepancy.

EXAMPLE 5 Production of Monoclonal Antibodies to S. uberis Streptokinase

Vaccination of mice

Purified streptokinase (10 μ l g) was mixed with Freund's incompleteadjuvant and injected subcutaneously into Balb/c mice. This procedurewas repeated after an interval of approximately 21 days.

Production of specific antibody was monitored 10 days after the secondinjection, as described below. Approximately 1 month following thesecond injection and 4 days prior to fusion of spleen cells (see below)antibody production was boosted by intravenous administration of 10 μgof purified streptokinase suspended in phosphate buffered saline (PBS;pH 7.2).

Production and cultivation of hybridoma cells

Spleens were removed from vaccinated mice and fusions carried accordingto standard methods (Galfre et al, 1977). Fused cells were resuspendedin HAT medium (RPMI 1640 (Gibco BRL, Life Technologies, Paisley, UK)containing 10% (v/v) foetal bovine serum, 0.1 mM hypoxanthine, 0.016 mMthymidine and 40 μM aminopterin Sigma, Poole, UK)). Cells were thendispensed at a concentration of 2×10 ⁶ cells/ml into 24-well clusterplates (1 ml/well) which had been seeded 24 h previously with 1 ml HATmedium containing 2×10⁴ murine macrophages. The plates were incubated at37° C. in the presence of 5% CO₂ until hybridoma colonies were clearlyvisible when samples of the supernatants were 2 0 tested for thepresence of streptokinase specific antibody. Colonies from which thesupernatant showed streptokinase specific antibody were grown up andsupernatants tested for anti-streptokinase activity as before, culturesshowing activity were cloned twice by limiting dilution.

Ascitic fluid was prepared by intra peritoneal (I.P.) injection ofBalb/c mice with 2-4×10⁶ cloned hybridoma cells. The mice had been givenan I.P. injection of 0.5 ml "Pristane"(a trademark of the AldrichChemical Co, Gillingham, UK) one week prior to this procedure.

Detection of streptokinase specific murine antibody

Purified, lyophilised streptokinase was redissolved in sodium carbonatebuffer (pH 9.6) at a concentration of 1 μg/ml and 100 μl was added towells of a flat-bottomed, 96-well micro-titre tray (Flow labs (Linbro),Virginia, USA). Streptokinase was allowed to bind to the wells for 18 hat 4 ° C. Unbound streptokinase was removed from the wells by washingwith excess ELISA-buffer (0.1 M sodium phosphate buffer, pH 7.2containing tween-20, 0.05% v/v). Antibody containing solutions wereadded to appropriate wells and allowed to react with the boundstreptokinase for 1 h at 37 ° C. Unbound antibody was removed by washingwith ELISA-buffer, as before. Bound antibody was detected by addition ofa murine immunoglobulin-specific antibody conjugated to horse radishperoxidase (HRP), incubation for 1 h at 37 ° C. and removal of unboundconjugate by washing in ELISA-buffer. This was followed by colorimetricdetection of the bound HRP by the addition of 100 μl of citrate buffer(24.3 ml of 0.05M citric acid and 25.7 ml of 0.1M Na₂ HPO₄) containing0-phenylaminediamine (0.34 mg/ml) and hydrogen peroxide (0.03%, v/v).Colour development was allowed to proceed for approximately 20 min andwas stopped by the addition of an equal volume of 1M sulphuric acid.

The colour was measured spectrophotometrically at a wavelength 15 of 492nm and the presence of specific antibody determined by comparison withthe colour development in control wells (wells which did not containstreptokinase and others to which the antibody containing solution hadnot been added).

Detection of streptokinase neutralising activity

Monoclonal antibodies (mAbs) were diluted in PBS (pH 7.2), and eachdilution mixed with an equal volume of streptokinase (0.5 μ g/ml). Themixtures were incubated at room temperature for 10 min after which 20 μl from each dilution of each mAb was placed into a well cut into a sheetof agarose (1% w/v in PBS) containing Oxoid skimmed milk (1%, w/v) and10--³ units/ml of bovine plasminogen (Sigma).

Wells containing mAb and streptokinase were compared to control wells towhich streptokinase and an equal volume of PBS had been added (positive)and those to which mAb alone had been added (negative). The ability ofmAbs to neutralise streptokinase is expressed as the lowestconcentration at which total inhibition of 0.5 μ g/ml was achieved(Table 1).

                  TABLE 1                                                         ______________________________________                                        Comparison of the neutralising activity of streptokinase                      specific monoclonal antibodies.                                               MONOCLONAL      INHIBITORY                                                    ANTIBODY        CONCENTRATION                                                 (Fusion No. Code No.)                                                                         (μg/ml)                                                    ______________________________________                                        F429.EC3        22                                                            F429.FG8        100                                                           F449.ED1        >140                                                          F449.DC2        23                                                            F450.DA3        >100                                                          ______________________________________                                    

EXAMPLE 6 Production of Polyclonal Antibodies to S. uberis Streptokinase

Vaccination of rabbit and detection of specific antibody

Purified streptokinase (50 μg) was mixed with Freund's incompleteadjuvant, and injected subcutaneously into a New Zealand white rabbit.This procedure was repeated on two further occasions at five and eightweeks after the initial injection. Production of specific antibody wasmonitored at various times after injection as described for murineantibody in Example 5, except that the antibody conjugated to horseradish peroxidase in this example was specific for rabbitimmunoglobulin.

Detection of streptokinase neutralising activity

The ability of immunoglobulin purified from the polyclonal serum usingimmobilised protein G (MAB TRAP Pharmacia, Uppsala, Sweden) toneutralise streptokinase activity was determined exactly as described inExample 5 for specific monoclonal antibodies.

The polyclonal antibody neutralised the activity ofS.uberisstreptokinase but not that from S.equisimilis which was obtainedfrom the Sigma Chemical Co. (Poole, Dorset UK)

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I claim:
 1. A vaccine for use to treat or prevent mastitis in a bovinespecies, comprising an immunologically effective amount of aplasminogen-activating streptokinase protein produced by Streptococcusuberis, and a pharmaceutically acceptable carrier.
 2. A vaccinecomposition for use to treat or prevent mastitis in a bovine species,comprising a plasminogen-activating streptokinase protein produced byStreptococcus uberis, and a pharmaceutically acceptable carrier.
 3. Amethod of treating or preventing mastitis in a bovine species,comprising vaccinating a cow with an immunologically effective amount ofa plasminogen-activating streptokinase protein produced by Streptococcusuberis, and a pharmaceutically acceptable carrier.
 4. An isolatedplasminogen-activating streptokinase protein produced by Streptococcusuberis,or an immunogenic fragment thereof.